JPS5988427A - Antihemophilic cold sediment low temperature sterilization - Google Patents

Abstract

A process for the pasteurization of antihemophilic cryoprecipitate (AHC) is described, wherein AHC is heated in the presence of calicum ions, an amino acid and a carbohydrate. The pasteurized AHC is used for the treatment of coagulation disturbances.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for pasteurizing anti-hemophilic cryoprecipitate (AHK) and the anti-hemophilic cryoprecipitate prepared thereby.

F■/vW concentrates from human plasma are used in the treatment of patients with hemophilia type A and patients with Fono-Willebrand syndrome (vW-syndrome). Currently, this treatment is used not only for immunization but also partly for autologous therapy.

Because of this, and because patients are treated for a long time throughout their lives, good products are needed.

This product is not the most purified and contains loaded proteins, which not only makes dissolution and application of such concentrates difficult, but also leads to sensitization to and associated with foreign proteins. Concomitant reactions pose a great risk to the patient, especially if they occur during self-treatment.

Factor ■Classification and nomenclature of molecules F■Bound Ag = FVI! AG = 1 lower prisoner (P, is [FV derived from ReLated AntigenJ)
l11 von Willebrand factor = F VN vW
F70 (derived from R15tocetinJ cofactor) F■Activity-F■C (Coagulation
)=F■C:AG (coagulation Antigen
Since the endogenous activity derived from enJ is suppressed by antibodies of the same series, it also refers to the C-antigen here.

The names in the box are based on international standards.

For the treatment of hemophilia type A, there is a highly pure, potent and well-balanced factor II concentrate. West German Patent Application Publication No. 2
From No. 916711, there is also No. II, which is safe for hepatitis.
Factor concentrates are known.

Unfortunately, however, factor Ⅰ concentrates do not exhibit optimal therapeutic efficacy in vitro studies even when they contain VCF ∙R:AG and FR∶CoF in addition to aFWI:C. Apparently, the in vitro test used to determine VW syndrome, the ristocetin cofactor test,
There is no correlation with bleeding time.

On the contrary, anti-hemophilic cryotherapy, ornamentation has a good effect on vW syndrome. The object of the present invention was therefore to produce AHKs that have good F■ and vW activities and are therefore safe against hepatitis.

The problem with solving this problem is that AHK, for example,
An example of denaturation and precipitation after heating at 60°C for 0 hours/L contains many types of residual proteins such as fibrinogen, fibrous, and cutin.

This problem was solved in principle as follows. That is, the AHK solution was heated in the presence of calcium ion, one type of amino I, and one type of monosaccharide, oligosaccharide, or sugar alcohol, but the sugar or sugar alcohol was added before the amino acid. .

The concentration of citrate should be below 10 mmol/l, the addition of calcium ions being still advantageous. And if this is not done, gelation will occur during pasteurization.

The subject of the invention is therefore an antihemophilic cryoprecipitate (AHK)
) solution contains calcium ions, one type of amino acid and one
A method for pasteurizing ARK, which is characterized in that it is heated in the presence of various monosaccharides or oligosaccharides or sugar alcohols.

Calcium ions range from a minimum of 0.2 mmol/l to a maximum of 100 mmol/l! , preferably in a concentration of 5 mmol/l. And CaCj? Preferably, it is supplied in the form of two solutions.

At least one of the amino acids glycine, α- or β-alanine, hydroxyproline, florin, curcumin, α-1β- or γ-aminobutyric acid, preferably glycine, is added.

The concentration reaches 1-6 mol/IIK.

Sucrose is preferably used as a carbohydrate at a concentration of 35 to 60.
．． ! 7/100m1! It is used as a solution.

For sufficient stabilization during pasteurization of AHK, the sucrose concentration is 601/1oor7e or higher, preferably 60
A solution of f//100- and 2 mol/l of glycine are required; if the concentration is lower than that (glycine 2 mol/l or less), coagulation will occur during heating and the F■:C activity will decrease. descend.

Furthermore, the complexing agent (complex forming agent) should be less than 10 mmol in citrate and less than 5 mmol in EDTA to be used during blood collection.

The observation that fibrinogen without complexing agent does not solidify upon heating but remains fibrillated allows the conclusion that the denaturation is attributable to the removal of calcium ions. Apparently fibrinogen binds calcium so tightly that low concentrations of complexing agent cannot remove it. This is significant as heating in the presence of 5 mmol citrate with the same stabilizer produces a clear AHK solution, whereas heating in the presence of 5 mmol EDTA produces a coagulum. I think that the.

The exact calcium ion concentration cannot be determined for this heterogeneous mixture. Therefore, fibrinogen, which constitutes a major part of this fraction, is also stabilized by calcium ions along with factor ①. Optimally, there is no complexing agent but calcium ions between 0.2 and 100 mmol/7
% should preferably be present at a concentration of 5 mmol/no (Table 1).

Heating is carried out until any hepatitis B virus that may be present in the AHK loses its infectivity. For this purpose, the temperature is 1 minute to 48 hours at 60-80°C, preferably 50-70°C.
℃ for 5 to 15 hours.

As a result of the study by polyacrylamide gel electrophoresis, AHK i'j obtained by the method of the present invention: does not contain fibrinogen polymer, fibrinogen, F) Kunko C,
F~IR: Due to its high content of CoF and fibronectin, it is the therapeutic agent of choice for the treatment of hemophilia and Fono-Willebrand syndrome, and is particularly effective in terms of safety for pasteurized hepatitis. It is. A further advantage resides in the good solubility of this product compared to other cold solutions, which can be attributed to the separation and removal of poorly soluble proteins and especially fibrinogen polymers.

The heated solution was diluted with a buffer solution so that the AHK was saturated with 2.2 mol/l of glycine and sodium chloride (12j; l/1
Prior to precipitation with 00ml), accompanying proteins and denatured proteins were separated and removed by precipitation.

The precipitated fraction was collected by centrifugation, lysed, dialyzed, and the factor Ⅰ activity was determined and adjusted to 6 m8 units/go. Then, after being clarified and sterilized by filtration, 100 d of solution is available! It was aliquoted into 250 d leaching flasks and vacuum lyophilized.

Experiments were conducted as summarized below.

Thermal stability of AHK with various stabilizers added 15 2
5 - Sedimentation, coagulation 3D
2 5 - Coagulation 60 2 5 - Clear 60 5 - Coagulation 60 0.5 5 - -
Hatondo Clear 60 1 5 - Clear 60 2” 5 - Clear 60 2
5 - Clear (5Q 2 10 - Coagulation 60 2
20 - Coagulation 60 2 5 -
Coagulation 60 2 - 10 -
Solidification 60 2 2.5 Clear 60 2 5 Clear 60 2 - - 10 Clear 60 2
- - 25 Clear saccharose concentration is insufficient to keep fibrinogen in solution.

Umbrella This glycine concentration ll''i-F■: is necessary in order to maintain the C activity during heating.

Table 2 shows the results of assaying the activity of AHK after heating 60 grams of untreated cryoprecipitate to 60° C. for 10 hours.

1 for separating and removing denatured products generated during heating.
．． 3 mol/I! 2.2 for precipitation with glycine, but also for removal of stabilizers and concentration of F/vW proteins.
Precipitation with mol/l of glycine and 129/100 of common salt can be carried out. The invention is demonstrated in more detail in the following examples.

EXAMPLE Preparation of pasteurized AHK Starting material: untreated external precipitate (Kuri no.) settled after separation of citrated plasma in a separator 250
．． ! i' (Brinkhous and Hernker
Mr. 75 “Handbook of Hemophilia”
G in liaJ 1975 edition Part 2, written by Mr. Poo1 [Cryoprecipit, ate: its
Preparat, 1onand C11nical
(Refer to UseJ) 1. 250 g of A/(OH)3 adsorbed chestnut was mixed with 0.1 mol/l Na at 37°C.
Dissolve in the required amount of C/solution to make a solution of 11, AA
'(OH) 315 suspended in 100 me of water 80
rn1. was added and stirred for 15 minutes. AA+(OH)'
3 was removed by centrifugation and discarded.

■ Add the required amount of Ca to the solution I by stabilization and pasteurization I, o o o
Add Cl2 aqueous solution, and the mixture becomes Ca++5 mmol/l.
to which saccharose 1,0OOJ9 was added at 37°C. Immediately after the sucrose was dissolved, 150 g of glycine was added and dissolved. 2
The pH was adjusted to 73 using normal NaOH and finally the solution was heated to 60° C. for 10 hours in a hot water bath.

After cooling, the solution obtained by protein isolation (■) and separation and removal of accompanying proteins and denatured products (■) was added to a citrate/NaCl solution (N
aC,eo, 06 mol/l and trisodium citrate 0.02 mol/11) 51! 648 g of glycine was added while stirring at 37°C, and after 15 minutes 1
It was cooled to 5°C and centrifuged.

The residue was discarded.

Collection of N,F'II/vW complex Glycine 449I was added to the above m amount of supernatant solution at 37°C with stirring, and then NaC Noah 98y was added with stirring.
CA! The concentration was brought to 1'21//100 Kyo. After all the fillers were dissolved, it was cooled to 15°C. 3.0O
OX,? A clear separation of the precipitate and the supernatant was achieved by centrifugation of the precipitate.
pH 7.3, and glycine 117100ml) 2
00m/! dissolved in. A solution of 350 ml was obtained.

■, The dialysis solution was dialyzed against 20 J of the same buffer as shown in ■, resulting in a solution with an electrical conductivity of 14 m5/fL400.
- became.

(2) Commercialization Following the above steps, ultrafiltration, clarification flow filtration, and sterilization filtration were carried out. Yield is pasteurized and 7'CAHK solution 5
00 ml were obtained and in some cases lyophilized under vacuum.

Below, pasteurized A prepared by the method according to the examples
The characterization of HK 30 is shown.

All 30 P゛■:C activities are within 35 degrees of the theoretical value based on the amount of Cryo as a starting material. However, the corresponding F■R:CoF value reaches 100chi.

Claims (1)

[Claims] 1) A solution of antihemophilic cryoprecipitate (AHK) is heated in the presence of calcium ions, one type of amino acid, and one type of monosaccharide, oligosaccharide, or sugar alcohol. A method for pasteurizing anti-hemophilic cryoprecipitates. 2) Calcium ion concentration is 0.2 to 100 mmol/
1. The processing method according to claim 1, characterized in that: l. 3) The amino acid is glycine, α- or β-alanine, hydroxyproline, proline, glutamine or α-1β- or γ-aminobutyric acid in a concentration of 1 to 3 mol/l The treatment method described in Section 1. 4) Carbohydrate concentration 35-60f7. 2. The processing method according to claim 1, wherein the saccharose is 1/100-. 5) The treatment method according to any one of claims 1 to 4, characterized in that heating is performed at a temperature of 50 to 70°C for 5 to 15 hours. 6) An anti-hemophilic cryoprecipitate prepared by the method according to any one of claims 1 to 5.